MEDICINE published: 18 November 2014doi: 10.3389/fmed.2014.00044

Pretargeted imaging and radioimmunotherapy of cancerusing antibodies and bioorthogonal chemistryFloor C. J. van de Watering1, Mark Rijpkema1, Marc Robillard 2,Wim J. G. Oyen1 and Otto C. Boerman1*1 Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, Netherlands2 Tagworks Pharmaceuticals, Eindhoven, Netherlands

Edited by:Jean-Pierre Pouget, INSERM, France

Reviewed by:Zhen Cheng, Stanford University, USALaurence Carroll, Imperial College, UK

*Correspondence:Otto C. Boerman, Department ofRadiology and Nuclear Medicine,Radboud University Medical Center,Geert Grooteplein Zuid 10, Nijmegen6525 GA, Netherlandse-mail: otto.boerman@radboudumc.nl

Selective delivery of radionuclides to tumors may be accomplished using a two-stepapproach, in which in the first step the tumor is pretargeted with an unlabeled antibodyconstruct and in the second step the tumor is targeted with a radiolabeled small molecule.This results in a more rapid clearance of the radioactivity from normal tissues due to thefast pharmacokinetics of the small molecule as compared to antibodies. In the last decade,several pretargeting approaches have been tested, which have shown improved tumor-to-background ratios and thus improved imaging and therapy as compared to directly labeledantibodies. In this review, we will discuss the strategies and applications in (pre-)clinicalstudies of pretargeting concepts based on the use of bispecific antibodies, which are capa-ble of binding to both a target antigen and a radiolabeled peptide. So far, three generationsof the bispecific antibody-based pretargeting approach have been studied.The first clinicalstudies have shown the feasibility and potential for these pretargeting systems to detectand treat tumor lesions. However, to fully integrate the pretargeting approach in clinic,further research should focus on the best regime and pretargeting protocol. Additionally,recent developments in the use of bioorthogonal chemistry for pretargeting of tumors sug-gest that this chemical pretargeting approach is an attractive alternative strategy for thedetection and treatment of tumor lesions.

Keywords: pretargeting, bispecific antibodies, tumor-associated antigen, radioimmunodetection, radioim-munotherapy

INTRODUCTIONThe use of radiolabeled monoclonal antibodies (mAbs) directedagainst tumor-associated antigens to visualize, characterize,and/or treat tumor lesions has been widely studied in preclinicaland clinical studies (1–3). After intravenous injection, the accumu-lation of the radiolabeled mAbs in the tumor is slow due to the slowextravasation and tissue penetration of intact antibodies. Variousphysiological barriers between the circulation and the tumor cellsurface, such as the vascular endothelium, the relatively large trans-port distances, and the high interstitial fluid pressure in tumors,prevent the rapid accretion of the antibodies in the tumor (4). Asa result, only a small fraction of the injected activity will localizein the tumor. Despite this inefficient targeting, in various clini-cal settings this approach can be effective: especially radiosensitivetumors, like Non-Hodgkin’s lymphomas,can respond to treatmentwith radiolabeled anti-CD20 antibodies (5). Effective radioim-munotherapy (RIT) requires high tumor uptake and rapid clear-ance of radioactivity from normal tissues. Various approaches havebeen investigated to improve the tumor targeting such as reducingthe circulatory half-life of the antibodies (4). Molecular engineer-ing techniques made it possible to produce antibody formats thatclear faster from the blood. However, this is the central dilemmain antibody targeting of tumors: on one hand, the high level ofmAb in the circulation is the driving force for the accumulation ofthe mAb in the tumor; on the other hand, the clearance from nor-mal tissues should be rapid. Therefore, innovative approaches are

needed to enhance tumor accumulation while limiting retention innormal tissues. Pretargeting is an approach to solve this problem.Several pretargeting systems have been developed including theavidin/streptavidin-biotin pretargeting system (4, 6) exploiting theextremely high binding affinity between biotin and (strept)avidin(K d= 4× 10−14 M) (7), the DNA-complementary DNA bindingpretargeting system, which relies on the high affinity interactionbetween complementary oligomers (8–11), pretargeting systemsbased on the use of bispecific antibodies (bsAb), and recently anovel pretargeting approach has been reported based on highlyselective bioorthogonal chemical reactions (12, 13). All pretar-geting systems are promising; however, each of these approacheshas its advantages and limitations. For the avidin/streptavidin,the most significant limitation is the immunogenicity of the pre-targeting agents: (strept)avidin being a protein that cannot behumanized. Additionally, the presence of endogenous biotin thatcould interfere with the system and the need to use a clearingagent to remove the residual antibody from the circulation beforeadministration of the radiolabeled biotin limit the application ofthis system (14). For the DNA-complementary DNA binding pre-targeting system, no immunogenic issues or complications dueto the presence of competing endogenous species are reported.However, the oligonucleotides need to be modified to preventrapid degradation by DNases and/or RNases (11). The pretarget-ing concept based on the use of bsAb directed against both a targetantigen and a radiolabeled hapten is carried out in the following

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van de Watering et al. Pretargeting of cancer

FIGURE 1 | Schematic overview of the pretargeting strategy. First, tumors are pretargeted with bispecific antibodies (bsAb). Secondly, small radiolabeledhapten peptide is i.v. injected and binds to the pretargeted bsAb at the tumor cells.

two steps. In the first step, the bsAb is injected. After the bsAbhas accumulated in the tumor due to the specific binding tothe tumor-associated antigen and cleared from the blood, theradiolabeled peptide carrying the hapten is administrated. Thissmall molecule will be trapped in the tumor by binding tothe bsAb or cleared rapidly from the body via the kidneys. Aschematic overview of this pretargeting strategy is shown inFigure 1. Depending on the radionuclide used this pretargetingapproach can be used to visualize, or treat tumor lesions, forexample, using 111In and 99mTc for single-photon emission com-puted tomography (SPECT) imaging, 68Ga or 18F for positronemission tomography (PET) imaging, or 131I, 90Y, and 177Lu forpretargeted radioimmunotherapy (PRIT). A significant advan-tage of this system over the biotin/avidin-based approaches isthat the primary pretargeting agent, the bsAb, can be human-ized to reduce its immunogenicity. Additionally, the lower affinityof the bsAb-hapten binding compared to the avidin-biotin bind-ing results in the rapid dissociation of the bsAb from the hap-ten in the circulation, thereby omitting the need for a clearingagent. In the past decade, several improvements on this systemhave been introduced, which resulted in a flexible universal andefficient pretargeting system. However, the non-covalent bind-ing between the radiolabeled hapten and the bispecific antibodyat the tumor cell surface limit the retention of the radiolabeledhapten-peptide in the tumor. Recent developments in the use ofbioorthogonal chemistry (cf. the rapid and selective formation ofa covalent bond between the pretargeting agent and the radiola-beled agent in vivo) are very promising and show the potential ofthis system for efficient pretargeting of tumors. Thus, this chemi-cal pretargeting system is an attractive alternative for bsAb-basedpretargeting.

In this review, we will focus on the pretargeting concept basedon the use of bsAb and additionally discuss the potential and

the limitations of this novel bioorthogonal chemistry pretargetingapproach.

BISPECIFIC ANTIBODY-BASED PRETARGETING SYSTEMSThe first studies on the use of a two-step system to target tumorswere described by Reardan et al. (15). This group used anti-bodies against radiolabeled EDTA. This study showed that byseparating the antibody tumor targeting from radionuclide tar-geting improved tumor-to-non-tumor ratios could be obtained(16). These antibodies had no affinity to the tumor and accumu-lated passively due to the enhanced permeability and retentioneffect in the tumor (4, 17, 18). For specific pretargeting of tumors,bsAb directed against both the tumor-associated antigens and theradiolabeled agent are required.

FIRST GENERATION; ANTI-TUMOR Fab′×ANTI-CHELATE-METAL FAB′

FRAGMENTSThe first generation of bsAb for specific pretargeting methods waschemically conjugated anti-tumor Fab′× anti-chelate-metal Fab′

fragments (6). Several preclinical studies revealed good tumoruptake (19, 20). Stickney et al. linked the sulfhydryl groups ofFab′ fragments of anti-CEA to those of an anti-benzyl-EDTA toform an F(ab′)2 bifunctional antibody (19). The potential of thisanti-CEA× anti-benzyl-EDTA F(ab′)2 was evaluated in mice bear-ing colon tumor xenografts. The anti-CEA× anti-benzyl-EDTAFab′× Fab′ was injected 24 h prior to the i.v. administration of111In-labeled benzyl-EDTA. One day later, the biodistributionrevealed a tumor uptake of 18.5%ID/g, whereas the blood levelwas 1.3%ID/g. A clinical study was performed to assess the useof this approach in patients with recurrent colon carcinoma. In14 patients, 20 of the 21 known tumor lesions were detected byscintigraphic imaging. Additionally, nine unknown lesions weredetected of which eight could be confirmed.

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SECOND GENERATION; THE USE OF A DIVALENT RADIOLABELEDHAPTEN-PEPTIDEThe pretargeting system using anti-tumor Fab′× anti-chelate-metal Fab′ fragments was based on monovalent binding tothe chelate–radiometal complex at the tumor. The stability ofsuch a complex is limited and rapid release of the radiola-beled chelate from the tumor may occur (21). Le Dousall et al.reported improved stability of the complex using divalent hapten-peptide (22). In BALB/c mice i.v. injection of bsAb directedagainst the lyb8.2 antigen and the hapten dinitrophenyl (DNP)followed by either the mono [N -ε-(2,4-dinitropheny1)-l-lysyl-diethylenetriaminepentaacetic acid (DNP-DTPA)] or divalent(di-DNP-DTPA).

The 125I-divalent DNP derivative exhibited a significantlyhigher binding as compared to the monovalent derivative to thepretargeted cells (for monovalent 23 vs. 65%ID/g for the divalentderivative). It was hypothesized that at the cell surface the divalenthapten could cross-link two bsAbs thereby stabilizing not only thebinding to the bsAb but also the bsAb binding to the tumor. Thisso-called affinity enhancement system (AES) improved tumor tar-geting and retention of the radiolabeled di-hapten-peptide (20,22). This AES was confirmed by different groups in several animalmodels, in which enhanced tumor uptake and improved reten-tion of the radiolabeled di-hapten-peptide was observed (23–28).For example, the use of a bsAb in combination with a diva-lent 111In-DTPA-peptide resulted in a tumor uptake of 3.5%ID/gwhereas the tumor uptake of the monovalent DTPA was 2.8%ID/gin mice with s.c. A375 melanoma xenograft. A pronounced AESeffect was observed in nude mice with RCC xenografts using bsAbdirected against the renal cell carcinoma-associated antigen G250,in combination with a divalent hapten-peptide, Phe–Lys(DTPA-111In)–Tyr–Lys(DTPA-111In), 111In-di-DTPA (16). At 1 h p.i. ofthe hapten, an almost 10-fold higher tumor uptake of 111In-di-DTPA (80%ID/g) was observed as compared to the monova-lent 111In-DTPA (9%ID/g; Figure 2). Moreover, the retention ofthe radiolabel in the tumor at 72 h p.i. was significantly higherfor the 111In-di-DTPA (93± 41%ID/g) compared to 111In-DTPA(0.9± 0.1%ID/g).

Based on the promising preclinical studies, a clinical trialwas performed to evaluate the pretargeting approach in patientswith primary colorectal cancers. In 11 patients with primary col-orectal carcinoma tumors, anti-CEA× anti-DTPA Fab′–Fab′ anti-body was injected followed 2–8 days later by 111In-labeled N -α-(In-DTPA)-tyrosyl-N -ε-(In-DTPA)-lysin (111In-di-DTPA) (29).For comparison, six patients with similar clinical status wereinjected with 111In-anti-CEA F(ab′)2. At 1–4 days p.i. of 111In-di-DTPA, the biodistribution results revealed a tumor uptakeof 1.8–17.5%ID/kg and were similar to the tumor uptake of111In-anti-CEA F(ab′)2 (5.5–30.2%ID/kg). However, the tumor-to-blood (T/B) and the tumor-to-liver (T/L) ratios were signifi-cantly improved in the pretargeting approach: 7.8 vs. 4.2 and 2.8vs. 0.8, respectively (29).

Besides using a pretargeting approach to detect tumor lesions,the pretargeting approach was also tested to treat tumor lesions.A clinical trial was conducted to estimate the dose delivered totumor targets and normal tissues using a pretargeting approachconsisting of bsAb anti-CEA× anti-DTPA and 131I-diDTPA (30).

FIGURE 2 | Biodistribution of 111In-diDTPA (A) and 111In-DTPA (B) at1–72 h after injection of the radioactivity in nude mice with s.c. NU-12tumors. Mice received 100 pmol of G250×DTIn1 bsAb i.v. and 3 days later7 pmol of 111In-DTPA or 111In-diDTPA. As a reference a separate set ofnude mice with s.c. NU-12 xenografts received the directly labeled antibody111In-DTPA-G250 (C). This research was originally published in CancerResearch by Boerman et al. (16).

In the clinical trial patients with recurrent medullary thyroid car-cinoma (MTC; five patients) and small-cell lung cancer (SCLC;five patients) were pretargeted with a bsAb anti-CEA× anti-DTPAFab′–Fab′ (0.1–0.3 mg/kg) and 4 days later 6 nmol (5.8–9.8 mCi)of 131I-di-DTPA was administrated. The patients were included

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based on the CEA expression as evaluated by immunohistochem-ical analysis of the biopsy of their primary tumor. The tumoruptake of 131I-di-DTPA and the activity dose to the tumor wassignificant higher in MTC (average uptake 0.116%ID/g; doseranged from 4.8 to 135 cGy/mCi) than in SCLC (average uptake0.009%ID/g; dose ranged from 1.9 to 8 cGy/mCi), indicating thatthe MTC is a more suitable tumor type for PRIT. The therapeu-tic efficacy and toxicity of PRIT with escalating doses of bsAbanti-CEA× anti-DTPA (20–50 mg) and 4 days later followed by131I-di-DTPA (1.48–3.7 GBq) was evaluated in 26 patients withrecurrent MTC (24). The dose-limiting toxicity was hematologicand an activity dose of 1.78 GBq/m2 could be injected safelyin this group of patients. The maximum tumor uptake rangedfrom 0.003 to 0.26%ID/g and the tumor doses ranged from 7.9to 500 Gy/MBq (2.94–184 cGy/mCi). From the 26 patients, 17patients were included to evaluate the tumor response. The max-imal tolerated dose (MTD) of 48 mCi/m2 was administrated to13 patients. A decrease in tumor mass, serum thyrocalcitonin,and CEA levels was observed in six of the patients receiving theMTD. The tumors that showed a response were generally small(maximum diameter of 37 mm).

Despite the promising (pre)clinical outcomes using a divalenthapten-peptide, the antibody dissociation rates were still higheras compared to the high affinity pretargeted avidin–biotin com-plex (K d= 10−14 M for biotin with streptavidin/avidin vs. 10−9 Mfor most antigen–antibody complexes) (7). It was suggested thatbsAb divalency to the primary target antigen might result inhigher tumor accretion by the pretargeted divalent peptide (21).A bsAb chemically prepared by coupling two Fab′ fragments,one with monovalent specificity to CEA and one to 111In-DTPA(Fab′× Fab′) was compared to bsAb with divalency to CEA. Twodivalent CEA bsAb coupled to a DTPA Fab′ fragment were com-posed either of the whole anti-CEA IgG (IgG× Fab′) or theanti-CEA F(ab′)2 fragment [F(ab′)2× Fab′). The antibody con-structs differ in molecular weight resulting in different clearancerates from the blood of which the fastest clearance is observedwith lowest molecular weight construct, Fab′× Fab′, followed byF(ab′)2× Fab′ and slowest for IgG× Fab′. The highest uptakeand retention in the tumor was achieved using the IgG× Fab′

bsAb conjugate. However, in a pretargeting strategy, the uptakeof the 111In-di-DTPA peptide (111In-IMP-192) was highest usingF(ab′)2× Fab′ conjugate followed by Fab′× Fab′ conjugate andlowest for IgG× Fab′. At the time of maximum tumor accretion,the IgG× Fab′ in the blood predominantly captured the injectedhapten. As a consequence, the three- to fourfold higher tumoraccumulation of the IgG× Fab′ could not be exploited in the pre-targeting approach. Consequently, to take full advantage of a highuptake and retention in the tumor using a long circulating bsAb,the accessibility of the bsAb remaining in the blood need to bereduced by using a clearing agent (21).

The flexibility of the bsAb pretargeting system is increased usingthe second generation anti-chelate bsAb as different radionuclidescan be used, for example, 111In (21, 29) for imaging and 131I fortreatment (24, 30). However, the binding affinity can be affectedby the chelated metal. For example, the affinity of the anti-DTPAantibody, designated 734, to various radiometal labeled DTPAvaries from 10−9 M for In-DTPA to 10−3 M for Ca-DTPA (31).

Additionally, the anti-(In)DTPA had a much lower affinity forDTPA with other radiometals such as 90Y or 177Lu. Therefore, itappeared that imaging radionuclides require different antibodiesdirected against the specific anti-chelate–metal complex than theradionuclides of therapeutic interest.

THIRD GENERATION; BISPECIFIC F(ab′)2ANTI-CEA×ANTI-HISTAMINE-SUCCINYL-GLYCINEA more universal/flexible pretargeting system was developed byusing an anti-hapten antibody not directed against the chelatedradiometal, but directed against the histamine-succinyl-glycine(HSG) hapten (22, 32, 33). The HSG hapten itself is not involvedin binding the radionuclide and thus other chelates suitable forbinding radionuclides can be added, for example, the HSG pep-tides can be conjugated with various chelating moieties suchas DTPA, DOTA, or N3S chelates for radiolabeling with In-111, Lu-177/Y-90, or Tc-99m/Re-188, respectively or with otheragents/moieties such as fluorophores for fluorescence imaging.Sharkey et al. showed the potential of this system by usingthe bispecific anti-CEA F(ab′)2× anti-HSG Fab in combinationwith HSG peptides labeled with either 99mTc or 188Re via anN3S chelate or with 111In, 177Lu, and 90Y via a DOTA moi-ety (32). In nude mice with human colon tumor xenografts,this trivalent anti-CEA× anti-HSG bsAb was injected 24–48 hprior to the i.v. administration of the divalent radiolabeled HSGpeptides, 111In-IMP241, 177Lu-IMP241, and 99mTc-IMP243. Thetumor uptake of radiolabeled HSG peptides after pretargetingwith the bsAb was significantly higher than with the peptidesalone (for 111In-IMP241 0.03± 0.02 vs. 2.54± 1.04%ID/g, for177Lu-IMP241 0.03± 0.01 vs. 2.59± 0.3%ID/g, and for 99mTc-IMP243 0.1± 0.02 vs. 7.36± 3.19%ID/g). The tumor-to-normaltissue ratio exceeded 8:1 within 3 h p.i. of the hapten-peptide.Consequently, this provides the opportunity to use the same HSGpeptide for different purposes depending on the radionuclideused, for example, using 111In and 99mTc-labeled HSG peptidefor SPECT imaging, 124I, 68Ga, or 18F-labeled HSG peptide forPET imaging or 131I, 90Y, and 177Lu-labeled HSG peptide forPRIT (32–38). Besides the flexibility of the system, Sharkey et al.showed that the hapten–peptide structure can alter the biodistrib-ution and clearance of the construct. The liver and kidneys uptakeof 99mTc-hapten-peptide consisting of a N3S chelate and DOTA(IMP245) was significantly lower compared to the 99mTc-hapten-peptide bearing only the N3S chelate whereas the tumor uptakeand retention did not change. Therefore, when less background isessential around the kidneys and/or urinary bladder, the preferablerenal elimination can be altered to a more hepatic elimination bymodifying the hapten-peptide construct.

Further improvements were obtained using a recombinant bis-pecific trivalent construct, hBS14, with bivalent CEA binding anda monovalent HSG binding (39). The trivalent antibody was pro-duced by myeloma cells transfected with hBS14-pdHL2 DNAvector and purified to near homogeneity in a single step usinga novel HSG-based affinity chromatography system. In nude micebearing a CEA-expressing GW-39 human colon tumor xenograftthe efficacy of the 125I-hBS14 (0.4 nmol) in combination with thebivalent 111In-HSG-hapten (0.04 nmol; 27 h interval), IMP241,was evaluated. At 3 h p.i., the 111In-IMP241 tumor uptake was

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FIGURE 3 | Schematic representation of how a trivalent bispecificantibody is formed by the DNL method. Component A links acysteine-modified dimerization and docking domain (DDD) to a Fab of ananti-tumor antibody, which results in spontaneous formation of componentA2. Component B links an anchoring domain to a Fab of an anti-haptenantibody. The AD is modified with cysteine on each end. AD will naturally“dock” with the DDD when components A and B are mixed, which bringsthe two molecules together in a well-defined orientation and also results indisulfide bonds across the two proteins. This research was originallypublished in Goldenberg et al. (42).

19.1± 8.7% ID/g and the blood levels were only 0.25± 0.08% ID/gcorresponding with a tumor-to-blood ratio of 83± 44% ID/g.

Technology that exploits fusing two hybridoma Ab-producingcells using quadroma technology did not produce adequate yieldsin mammalian cell cultures. Therefore, a new strategy capable ofpreparing a trivalent bsAb was developed. Humanized trivalentFab bsAb constructs were produced at a higher yield by the dock-and-lock (DNL) technology. The DNL technology is based on thenatural association between protein kinase A (PKA; cyclic AMP-dependent protein kinase) and A-kinase anchoring proteins. Theregulatory subunit of PKA and the anchoring domain of the inter-active A-kinase anchoring protein are both attached to a biologicalentity. The dimeric sequence attached to the anti-tumor F(ab)2 hashigh affinity for the sequence attached to the anti-hapten Fab, theso-called docking. The stabilization of the trivalent bispecific anti-body construct, the so-called locking, is secured by the placementof cysteine residues on four locations within the construct result-ing in the formation of disulfide bridges (40–42). A schematicoverview of the DNL technology is shown in Figure 3.

Using the DNL technology, several recombinant humanizedbsAbs with a divalent antigen specificity and with a monovalentspecificity to HSG were produced including an anti-CEA bsAb(TF2) (43, 44), an anti-Trop2 (TF12) (45), an anti-CD20 (46), andan anti-MUC1 (47) bsAb.

An effective detection and treatment of tumor lesions using apretargeting system is best illustrated by the work done on TF2.The ability to detect small micrometastatic human colon cancernodels (<0.3 mm in diameter) in the lungs of nude mice using

this system was evaluated and compared to 18F-FDG-PET imag-ing and to TF6, an irrelevant anti-CD22-based bsAb. The tumorswere first pretargeted with TF2 and 21–24 h later the 124I or 131Iradiolabeled hapten-peptides were injected at a bsAb:hapten molarratio of 10:1. The TF2-pretargeted tumors in the lungs could belocalized at 1.5 h p.i. of radiolabeled HSG peptide whereas thepeptide alone, TF6, or 18F-FDG failed. This showed the highpotential of the system for sensitive and specific imaging of tumorlesions.

The specificity of the TF2 based pretargeting system was fur-ther evaluated using di-HSG peptides radiolabeled with 68Ga,a positron emitting radionuclide with a more suitable half-life(68 min) for pretargeted imaging purposes than 124I (4.2 days)(48). Nude mice were s.c. implanted with CEA-expressing LS174Thuman colonic tumor, a CEA-negative tumor, or an inflamma-tion was induced in the thigh muscle. The mice were first i.v.injected with bsAb anti-CEA× anti-HSG and after 16 h followedby 68Ga-IMP288. At 1 h p.i., a high specific uptake of 68Ga-IMP288 was observed in the tumor (10.7± 3.6%ID/g), whereasthe uptake of 68Ga-IMP288 in normal tissues, in CEA-negativetumors and inflamed muscle was low (Figure 4). In contrast,18F-FDG localized efficiently in the tumor, however, also in theinflamed tissue and in a number of normal tissues includingliver, spleen, and intestines. The specificity of the pretargetedimmuno-PET using TF2 and 68Ga-IMP288 was confirmed in micewith small intraperitoneal xenografts (49). All intra-abdominaltumors lesions >~4 mm and even a few lesions as small as~2 mm were detected. In line with the previous study, the 18F-FDG uptake in all the present tumors was sufficient, however,also an uptake was observed in various normal tissues such as theintestines.

To evaluate the use of the TF2 and 177Lu-IMP288 in PRIT,the therapeutic efficacy and toxicity of the system was evalu-ated in mice with s.c. LS174T tumors (50). The median survivalof mice treated with 1, 2, or 3 cycles of PRIT was 24, 45, and65 days, respectively, whereas in the untreated mice the mediansurvival was 13 days. Additionally, PRIT effectively delayed thetumor growth with limited hematologic toxicity, indicating thatPRIT using TF2 and 177Lu-IMP288 might be an effective treatmentagainst colon cancer. Furthermore, the biodistribution of 111In-IMP288 and 177Lu-IMP288 in mice with intraperitoneal LS174Ttumors was identical as observed by ex vivo counting as wellas by pretargeted immuno-SPECT imaging (51). This indicatesthat pretargeted immuno-SPECT with TF2 and 111In-IMP288 canbe used for the non-invasive monitoring of the therapeutic effi-cacy of PRIT with TF2 and 177Lu-IMP288 in mice with LS174Tlesions.

The preclinical pretargeting studies using TF2 in combinationwith radiolabeled di-HSG peptides indicated that PRIT can inducetumor growth inhibition. Therefore in the feasibility, safety andtherapeutic efficacy of TF2/Lu-177-IMP288 for the treatment ofCEA-expressing tumor lesions was investigated in a first-in-manphase I study in patients with advanced colorectal carcinoma. Fourdose schedules in cohorts of five patients were evaluated (52).First, the effect of the time interval between administration ofTF2 (75 mg) and IMP288 (100 µg) was evaluated: 5 days (cohort1) and 1 day (cohort 2). Additionally, the effect of a higher bsAb

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FIGURE 4 | PET/CT images of a BALB/c nude mouse with a s.c. LS174Ttumor on the right hind leg (arrow) and an inflammation in the left thighmuscle (arrowhead), which received 18F-FDG and, 1 day later, 6.0 nmol TF2and 68Ga-IMP288 (0.25 nmol) with a 16-h interval. The animal was imaged1 h after 18F-FDG and 68Ga-IMP288 injections. The panel shows thethree-dimensional volume rendering the pretargeted immuno-PET scan (A)and the FDG-PET scan (B), and the transverse sections of the tumor regionof the pretargeted immuno-PET scan (C) and the FDG-PET scan (D). Thisresearch was originally published in Schoffelen et al. (48, 50).

dose (150 mg TF2, 1-day interval, 100 µg IMP288, cohort 3) anda lower IMP288 dose (75 mg TF2, 1-day interval, 25 µg IMP288,cohort 4) was evaluated. Reducing the time interval and loweringthe IMP288 dose resulted in improved tumor targeting. Althoughit is reported that higher bsAb doses results in improved tumoruptake of the radiolabeled hapten (48, 50), no such observationswere found using twofold increase of TF2 dose. Co-localizationof almost all 18F-FDG positive tumors and hapten-peptide withSPECT was observed (Figure 5). Using a high TF2 dose and alow IMP288 peptide dose rapid targeting of tumors was observed,although wash-out of the tumor was observed after 24 h. Thepatients could tolerate 7.4 GBq of 177Lu-IMP288 without expe-riencing dose-limited toxicity, even though some patients (8 outof 20 patients) experienced some level of hematologic toxicity. TheTF2 bsAb induced human anti-human antibodies (HAHA) in 11out of 21 patients. Most likely, the best PRIT regime will be a frac-tionated multi-dose treatment regimen (e.g., multiple cycles) asobserved in preclinical pretargeting experiments (50) and recentclinical trials using 90Y-labeled antibodies in combination withgemcitabine (53). However, further clinical studies regarding thepretargeting conditions and protocol are needed.

FIGURE 5 |The SPECT/CT image (A), acquired 24 h after injection of111In-IMP288 (185 MBq, 25 µg), pretargeted with 75 mgTF2 (1-dayinterval), in a 38-year-old patient (cohort 4), shows very clear tumortargeting of an axillary lymph-node metastasis, with very lowconcentrations of radioactivity in normal tissues. Correspondingcontrast-enhanced CT scan and a fused FDG-PET/CT scan are shown[(B,C), respectively]. The primary colon tumor (50 cm ab ano) also showshighly specific tumor targeting in the SPECT image (D), confirmed by theCT scan and FDG-PET/CT [(E,F), respectively], with non-specific FDGuptake in the ascending colon. This research was originally published inSchoffelen et al. (52).

A DIFFERENT APPROACH; PRETARGETING BASED ONBIOORTHOGONAL CHEMISTRYThe combination of chemistry and biology has resulted in manyinnovations and has contributed to our understanding of manybiological processes. Nevertheless, many biomolecules includinglipids,glycans,and nucleic acids as well as various posttranslationalmodifications cannot be monitored with genetically encodedreporters as manipulations can interfere with the structure andfunction of the molecules or the molecules are not geneticallyencoded. Therefore, new methods were investigated to covalentlymodify biomolecules in vivo. This has led to the discovery ofbioorthogonal reactions to detect, track, or visualize various bio-molecules. Using bioorthogonal chemistry, as defined by Bertozziet al., reactions can occur inside a living organism, which do notinterfere or cross-react with naturally occurring functionalities, arereactive under mild and physiological conditions and should notinduce cellular toxicity (54). Over the years, several bioorthogo-nal chemical ligation strategies have been developed including thetetrazine ligation, Staudinger ligation, oxime/hydrazone forma-tion from aldehydes and ketones, and quadricyclane ligation [forreviews on these bioorthogonal chemical reactions see in Ref. (13,55, 56)]. The archetypical bioorthogonal reaction is the copper-catalyzed 1,3-dipolar Huisgen“click”cycloaddition between azidesand alkynes (55). Using this click chemistry, units are joined withheteroatom links (C-X-C) in a modular, rapid, and easy mannerwithout the generation of toxic byproducts (57). The selectivity

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and flexibility as well as the bioorthogonality and speed of clickchemistry make it an ideal method for the creation of radiophar-maceuticals, especially 18F-radiolabeled peptides (58). The bind-ing of a radiolabeled probe to the tumor-targeted antibody mightalso be obtained using bioorthogonal reactions. In general, thischemical approach of pretargeting is less likely to be immunogenicand could provide a universal approach for tagging and in vivotracking of Abs. However, the need for a copper catalyst in couplingof azide and terminal alkyne to generate a triazole limits the use ofthe 1,3-Huisgen cycloaddition reaction in biological systems due tothe toxicity of copper in vivo. Various bioorthogonal reactions havebeen developed that do not need the presence of a Cu catalyst. Forexample, copper-free click chemistry can be achieved via the reliefof strain (strain-promoted azide-alkyne cycloadditions, SPAAC).Alternatively, Blackman et al. reported the very fast reaction kinet-ics and in vitro bioorthogonality of the inverse-electron-demandDiels–Alder (IEDDA) reaction between trans-cyclooctene (TCO)and electron deficient tetrazines (59). Subsequently, Devaraj et al.tagged the anti-EGFR antibody cetuximab with trans-cyclooctenesuccinimidyl carbonate and combined with a fluorescent tetrazineprobe was able to target A549 cancer cells in serum at 37°C (60). Aschematic overview depicting the use of TCO-modified mAb andradiolabeled tetrazine to (pre)target tumors through the IEDDAreaction is shown in Figure 6. The first in vivo proof-of-conceptstudy by Rossin et al. using a chemical pretargeting approach basedon the Diels–Alder components demonstrated that the systemcould manage the more demanding conditions in vivo, includ-ing low reagent concentrations and short reaction time, and theprolonged residence time and required in vivo stability for theTCO tag (61). In mice bearing a colon-cancer xenografts, TCO-modified anti-TAG72 mAb CC49 (CC49-TCO) was administratedand 1 day later followed by 111In-labeled-DOTA-tetrazine (111In-tetrazine) at a 1:25 molar ratio. Three hours p.i. of 111In-tetrazine,SPECT imaging clearly delineated the tumor with a tumor uptakeof 4.2%ID/g and a tumor-to-muscle (T/M) ratio of 13.1, whereasthe tumor uptake of 111In-tetrazine and tumor-to-muscle ratiopretargeted with unmodified CC49 or an irrelevant TCO-modified

Ab were 0.3 and 0.5%ID/g or 1.0 and 2.1%ID/g, respectively(Figure 7).

Zeglis et al. demonstrated that the in vivo click method-ology is able to delineate tumors with PET (62). Nude micewith s.c. SW1222 colorectal cancer xenografts were i.v. adminis-trated with TCO-modified A33 Ab (100 µg) followed 24 h later by64Cu-NOTA-tetrazine (10.2–12.0 MBq) or with the directly radi-olabeled Ab 64Cu-NOTA-A33 (10.2–12 MBq) or 89Zr-DFO-A33(10.2–12.0 MBq). Despite the higher tumor accumulation of thedirectly labeled antibodies at 24 h p.i, the pretargeting approachresulted in comparable PET images and tumor-to-muscle ratios(Figure 8).The dose delivered to normal tissues using this pretar-geting approach was calculated, indicating that the non-targetedtissues received a significant lower dose when a pretargetingapproach (0.0124 mSv/MBq) was used compared to the directlylabeled Abs, 64Cu-NOTA-A33, and 89Zr-DFO-A33 (0.0359 and0.4162 mSv/MBq, respectively).

The fast reaction kinetics [k2= 13,090 M−1s−1 (60)] of theIEDDA reaction between the trans-cycloctene and tetrazines arevery promising, however, are still significantly lower comparedto the association constants of the non-covalent high affinityinteractions used in humans (5× 105 up to 7.5× 107 M−1s−1).In addition, Rossin et al. reported that the TCO can be deac-tivated through isomerization to the unreactive cis-cyclooctene(CCO) isomer via copper-containing proteins (e.g., transcuprein,mouse serum albumin, ceruloplasmin) (61). TCO tags that arelinked to the antibody through an axial substituent afforded amarked increase of the reactivity (61). In conjunction, the sta-bility of the TCO tag could be improved by removal of the PEGlinker between the TCO and the lysine residue on the Ab, increas-ing the steric hindrance on the TCO thus hampering interactionwith serum protein-bound copper. The increased reactivity (up tok2= 2.7× 105 M−1s−1 in PBS (61) and improved stability of theIEDDA between a tetrazine and TCO-tagged antibody resultedin an improved tumor-to-blood ratio (61). Devaraj et al. (64)reported that the in vivo reaction yield of a moderately reactiveIEDDA system (k2= 6× 103 M−1s−1 in PBS) could be improved

FIGURE 6 | Schematic overview of tumor pretargeting by using the inverse-electron-demand Diels–Alder reaction. This research was originally publishedin Rossin et al. (60).

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by altering the pharmacokinetics of the radiolabeled probe bypolymer conjugation (64). In mice with LS174T xenografts TCO-tagged A33 antibody (30 µg) was administrated and followed 24 hlater by the 18F-labeled, dextran-based tetrazine probe (30 µg;150 µCi). PET imaging 3 h p.i. of the 18F-labeled tracer revealed a

FIGURE 7 | SPECT/CT imaging of mice bearing colon carcinomaxenografts: posterior projections of mice preinjected with (A)CC49-TCO followed 1 day later by 111In-DOTA-tetrazine (1:25, 42 MBq),(B) CC49 followed 1 day later by 111In-DOTA-tetrazine (1:25, 20 MBq), (C)irrelevant Ab (Rtx-TCO; 100 µg) followed 1 day later by111In-DOTA-tetrazine (1:25, 50 MBq), (D–F) single transverse slices(2 mm) passing through the tumors in (A–C). This research was originallypublished in Rossin et al. (60).

significant higher tumor accumulation in TCO-tagged antibodypretargeted mice compared to mice pretargeted with Ab lack-ing TCO. However, the tumor-to-blood ratio still remained lowdue to the relative high amount of free-circulating CC49-TCO.For effective tumor targeting, a clearing agent was developedthat could clear the TCO-tagged antibody from the circulationprior to administration of the radiolabeled tetrazine (61). In micewith LS174T tumor xenografts, 125I-CC49-TCO was injected afterwhich a single or double dose (30 h or 30 and 48 h after mAb injec-tion) of the clearing agent, followed 2 h later by 177Lu-tetrazine.Three hours p.i., the blood level of 125I-CC49-TCO was lowestafter the double dose of clearing agent (0.19± 0.04%ID/g), fol-lowed by the single dose (1.16± 0.43%ID/g) and highest in thegroup without clearing agent injection (8.47± 4.12%ID/g). Thebiodistribution of 177Lu-tetrazine showed that the eliminationof the free CC49-TCO in combination with a reduced amountof tetrazine (from 17 to 6.7 nmol) significantly improved thetumor uptake from 3.12± 0.87%ID/g (60) to 7.45± 1.46%ID/gfor the single dose and to 6.13± 1.09%ID/g for the double doseof clearing agent. Both single as well as the double dose approachsignificantly increased the tumor-to-muscle and tumor-to-bloodratios compared to the previous approach without clearing agent.In fact, a 125-fold improvement of the tumor-to-blood ratio at3 h after tetrazine injection was achieved with a double dose ofclearing agent. Corresponding mouse dosimetry experiments sug-gested that at MTD for bone marrow (dose-limiting organ forboth approaches), this should allow for an eightfold higher tumordose than is possible with non-pretargeted RIT. The estimateddose to the tumor in mice with directly 177Lu-labeled CC49 issignificantly higher compared to the pretargeted approach usingCC49-TCO and 177Lu-Tz (9573 vs. 479 mGy/MBq). However, alsothe normal tissues receive high doses, for example, the bone mar-row receive 1136 mGy/MBq, whereas in the pretargeting approachthe bone marrow dose is 7 mGy/MBq. Subsequently, this sys-tem was further improved by modifying the pharmacokineticsof the tagged antibody and by increasing the stability of the tag.The use of a more hydrophilic tag with an increased in vivo tagstability (t 1/2= 10 days) afforded a slower clearing CC49-TCOconjugate with increased tumor targeting, resulting in a 50%increased tumor uptake and T/NT ratios of the tetrazine probe(65). So far, the reaction of TCO with 1,2,4,5-tetrazines is the

FIGURE 8 | PET images of 64Cu-Tz-Bn-NOTA/A33-TCO pretargeting strategy, 64Cu-NOTA-A33 and 89Zr-DFO-A33.Transverse (top) and coronal (bottom)planar images intersect the center of the tumors. This research was originally published in Zeglis et al. (63).

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fastest bioorthogonal reaction (61). While others were unsuccess-ful in enlisting the slower SPAAC for antibody-based pretargetingin mice (62), Lee et al. reported a pretargeting system consistingof aza-dibenzocyclooctynes modified mesoporous silica nanopar-ticles in combination with 18F-azide and achieved tumor uptakein mice, possibly due to the high loading capacity of the meso-porous particles (66). Further work is required to establish whetherthe fastest cyclooctynes enable efficient SPAAC-based pretargetingwith antibodies as well.

CONCLUSIONClinical studies revealed that the pretargeting approach based onbsAb directed against a tumor-associated antigen and against HSGin combination with radiolabeled (divalent) peptides contain-ing the HSG residue resulted in high contrast images. There-fore, the use of this pretargeting system is very promising fornon-invasive imaging of tumors before, during, and after ther-apy. Additionally, pretargeted RIT using this system is capableto inhibit tumor growth with minor toxicity. However, to fullydevelop this pretargeting approach in the clinic research shouldfocus on the best regime and protocol. Although several com-parative studies between a pretargeting approach and directlyradiolabeled antibodies have been performed, further quantita-tive comparative studies are needed to evaluate the potential of thepretargeted imaging and therapy. Furthermore, preclinical stud-ies using bioorthogonal chemical pretargeting based on the inverseDiels–Alder cycloaddition revealed that tumors can be imaged andtreated with this new approach. This approach is still being fur-ther optimized and clinical studies are warranted to determine thepotential of this new strategy.

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van de Watering et al. Pretargeting of cancer

Conflict of Interest Statement: The authors declare that the research was conductedin the absence of any commercial or financial relationships that could be construedas a potential conflict of interest.

Received: 04 September 2014; accepted: 22 October 2014; published online: 18November 2014.Citation: van de Watering FCJ, Rijpkema M, Robillard M, Oyen WJG and BoermanOC (2014) Pretargeted imaging and radioimmunotherapy of cancer using antibodiesand bioorthogonal chemistry. Front. Med. 1:44. doi: 10.3389/fmed.2014.00044

This article was submitted to Nuclear Medicine, a section of the journal Frontiers inMedicine.Copyright © 2014 van de Watering, Rijpkema, Robillard, Oyen and Boerman. Thisis an open-access article distributed under the terms of the Creative Commons Attri-bution License (CC BY). The use, distribution or reproduction in other forums ispermitted, provided the original author(s) or licensor are credited and that the origi-nal publication in this journal is cited, in accordance with accepted academic practice.No use, distribution or reproduction is permitted which does not comply with theseterms.

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